Excessive mucus production is an important contributor to morbidity and mortality in common lung diseases, including asthma and COPD. We recently discovered that the protein anterior gradient homolog 2 (AGR2) plays an essential role in mucus production. AGR2 is a member of the protein disulfide isomerase (PDI) family that is found in the endoplasmic reticulum of mucus-producing cells. PDI family members assist in the folding and assembly of secreted proteins by catalyzing the rearrangement of cysteine disulfide bonds. There are 19 known mammalian PDIs but little is known about the specific roles of individual PDIs. Our data indicate that AGR2 has a specialized role in processing of mucins, the enormous cysteine-rich glycoproteins that are responsible for the characteristic viscoelastic properties of mucus. We produced Agr2-/- mice and found that these mice developed normally but were completely devoid of intestinal mucus and the major intestinal mucin MUC2. In a mouse model of asthma, production of the major airway mucin MUC5AC was reduced but not abolished in Agr2-/- mice. Preliminary studies indicate that MUC5AC is a substrate for AGR2 and for AGR3, a closely related PDI that is highly expressed in airway epithelial cells. We hypothesize that AGR3, like AGR2, plays a role in airway mucus production and that blocking expression or function of both AGR2 and AGR3 will dramatically reduce or completely prevent airway mucus production. Further studies are required to test this hypothesis and to determine whether AGR2 and AGR3 play a direct role in processing of any other proteins. We have three specific aims designed to provide a detailed understanding of the functions of AGR2 and AGR3 at the molecular, cellular, and organismal levels. In Aim 1, we will analyze AGR2 and AGR3 substrate binding specificity. In Aim 2, we will examine effects of AGR2 and AGR3-deficiency in the mouse airway. In Aim 3, we will analyze functions of AGR2 and AGR3 in human airway epithelial cells. The proposed studies will advance this novel area of investigation by providing fundamental information about the functions of AGR2 and AGR3 and by determining whether these unusual PDIs are promising therapeutic targets. PUBLIC HEALTH RELEVANCE: Excessive mucus production contributes to morbidity and mortality in common lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). We discovered a protein that is necessary for mucus production. This project will help us understand mucus production better and might lead to new treatments.